The present invention relates to the creation of DNA sequences coated with papillomavirus capsid proteins L1 and L2. In particular, the present invention relates to the packaging of papillomaviral genomic DNAs and thereby the efficient generation of infectious papillomavirus particles containing wild type or modified viral genomic DNAs.
Papillomaviruses are non-enveloped, double-stranded DNA viruses with ˜8 kb, circular genomes, 55 nm spherical capsid coats, wide distribution in higher vertebrates and tight species specificity. Human papillomaviruses (HPVs), of which there are over 100 genotypes, infect and replicate in cutaneous or mucosal epithelia, inducing benign lesions including warts that are self-limiting and normally regress over time. A subset of the mucosotropic HPVs (HPV), termed the high risk genotypes such as HPV16, 18, and 31, are causally associated with anogenital cancers, including nearly if not all cervical carcinoma, a leading cause of death by cancer among women worldwide. In addition high risk HPVs, in particular HPV16, are associated with 20-30% of head and neck cancers, though here an etiological role has yet to be clearly established.
The HPV life cycle is tightly linked to epithelial differentiation. HPVs initially infect cells of the poorly differentiated, proliferative, basal compartment of stratified epithelia. Here the viral genome sets up residence as a low copy nuclear plasmid and a subset of viral genes, termed the early genes are expressed at low levels. No progeny virus is made in basal cells. However, as infected basal cells grow and divide and daughter cells migrate into the suprabasal compartment to undergo terminal differentiation, the productive stage of the viral life cycle is initiated. Here the virus reprograms suprabasal cells to support the amplification of the viral genome to high copy number, the viral structural genes encoding the major and minor capsid proteins, L1 and L2, respectively are expressed, progeny virions are assembled and these virions then are released into the environment from the most superficial layers of the epithelia. The requirement for terminal differentiation of epithelial cells to support the productive stage of the viral life cycle precludes obtaining infectious virus particles from conventional cell culture. Consequently, the only prior methods capable of producing infectious papillomavirus virions were organotypic culture, a process by which small quantities of artificial skin can be produced in cell culture, or alternatively the use of xenografts in immunodeficient mice. However, these methods are technically demanding, time-consuming, variable, produce only relatively low virus yields, and require access to epithelial cell populations or human tissue in which the viral genotype of interest persists as a nuclear plasmid. These limitations have severely restricted the availability of infectious human as well as some animal papillomaviruses for basic, preclinical and clinical research.
Recently, Buck and Schiller developed a new approach to papillomavirus packaging in which reporter plasmids were encapsidated into bovine papillomaviral capsid proteins expressed in transiently transfected mammalian cells (J. Virol. 78:751-757, 2004). High levels of papillomavirus L1 and L2 capsid proteins were expressed from codon-optimized synthetic genes in 293TT human embryonic kidney cells, which stably express SV40 large T antigen to enhance replication of SV40 origin-containing plasmids. When cotransfected into 293TT cells with L1- and L2-expression plasmids, target plasmids of less than 6 kb were efficiently encapsidated into the resulting papillomavirus capsids. This method allowed for the differentiation-independent generation of virus-like transducing particles. However, Buck, et al. concluded that intracellular packaging of target plasmids into these papillomavirus-like transducing particles by this approach was limited by a strong size discrimination to target plasmids of 6 kb or less, far under the natural viral genome size of approximately 8 kb.
What remained in need is a method that permits for the efficient encapsidation of full-length and near-full length papillomaviral genomes into the papillomaviral capsids. This would allow for the efficient generation of infectious papillomavirus particles.